Telomerase and the consequencesTelomerase and the consequences of telomere dysfunction

Carol W. Greider

Setting the stage: telomere function isSetting the stage: telomere function is conserved

Ori

Marker

Linear plasmidAdd Tetrahymena telomeresT f t

TelomereTelomere MarkerOri

Transform yeast

Cell May 1982

Setting the stage: telomere elongation inSetting the stage: telomere elongation in trypanosomes

Nature June 1983

Setting the stage: Yeast sequences addedSetting the stage: Yeast sequences added onto Tetrahymena telomeres

Telomere MarkerOri

Maintain linearplasmid in yeast

Remove right telomereRemove right telomereAdd yeast genomic DNAYeast telomeres stabilizeslinear plasmid

Nature July 1984

Two different models for elongationTwo different models for elongation

RECOMBINATION DE NOVO ADDITION

3’5’

3’5’

3’5’

3’5’

Strand invasion

5

Extension of �3’ end

5

Extension of �3’ end

5

Elongation

3 end

Repair synthesis

3 end

Repair synthesis

Resolution &repair synthesis

Net telomereelongation

p y

Net telomereelongationNet telomereelongationelongationelongation

The hunt forThe hunt for telomerase

Telomerase activityTelomerase activityidentified

Greider & Blackburn Cell 1985

Identification of the RNA componentIdentification of the RNA component

Greider & Blackburn Cell 1987

Telomerase uses RNA template to add TTGGGG repeats

Greider & Blackburn Nature 1989

What happens without telomerase?What happens without telomerase?

Greider &Greider &Harley 1988

Telomere shortening in cellularTelomere shortening in cellular senescence

Nature May 1990

Telomeres are shorter in cancer cellsTelomeres are shorter in cancer cells

Nature 1990Mol Cell Biol. 1990

Telomerase protein component identified

Science 1997

TERT transfection extends the lifespan f h llof human cells

Science 1998

Using genetics to examine the role ofUsing genetics to examine the role of telomerase in mammals

Maria BlascoMaria Blasco

Telomerase knockout mouseTelomerase knockout mouse

mTR+ / - mTR+ / -

mTR+ / + mTR+ / mTR / G1 G1mTR+ / + mTR+ / - mTR- / - G1 G1

G2 G2

G3 G3

G4 G4

G5 G5

Cell deathorG5 G5

G6 G6

Senescence

Blasco et al. Cell 1997

Quantiative analysis of telomere lengthQuantiative analysis of telomere length

Telomeres shorten progressively inTelomeres shorten progressively in telomerase-null mice

mTR+ / - mTR+ / -

TR / (G1) G1mTR- / - (G1) G1

G2 G2

G3 G3

G4 G4

G5 G5G5 G5

G6

Blasco et al. Cell 1997

Loss of telomere sequence leads to lossLoss of telomere sequence leads to loss of telomere function

WT KO-G2

KO-G4 KO-G6

The consequences of short telomeresThe consequences of short telomeres

mTR+ / - mTR+ / -

mTR+ / + mTR+ / mTR / G1 G1mTR+ / + mTR+ / - mTR- / - G1 G1

G2 G2

G3 G3

G4 G4

G5 G5

Cell deathorG5 G5

G6 G6

Senescence

Mike HemannMike Hemann

Mice with 1/2 short and 1/2 long telomeresMice with 1/2 short and 1/2 long telomeres show telomere dysfunction

mTR+ / - mTR- / - G6

mTR- / - (iG7)mTR / (iG7)

mTR+ / - (iG7)

Hemann et al. Cell 2001

The shortest telomere triggers cell death

100

The shortest telomere triggers cell death

10

20

30

40

50

60

70

80

90

mTR+/- MEAN 44

0

10

40

50

60

70

80

90

100

mTR-/-G6 MEAN 24

0

10

20

30

mTR-/- 100

120

140

mTR /(iG7)

140

MEAN 32

0

20

40

60

80

mTR+/-(iG7)

20

40

60

80

100

120

MEAN 33

0

Hemann et al. Cell 2001

Short telomeres induce a DNAShort telomeres induce a DNA damage response

TelomereDNA

Double strand break Telomere shortening

p53

Apoptosis

Senescence

Telomerase is required for cells thatTelomerase is required for cells that must divide many times

CANCER CELLS STEM CELLS

Short telomeres supress tumorigenesisShort telomeres supress tumorigenesis

ff f

S ll i D G l S t l N t

Cancer modelEffect of short telomeres Reference

Squameous cell carcinoma (DMBA)

Colon cancer (APCmin)

Decrease

Decrease (apoptosis)

Gonzalez-Suarez et al. Nat Gen. 2000

Rudolph et al. Nat Gen. 2001(APCmin)

Lymphoma& fibrosarcoma(INK4a/Arf -/-)

T cell lymphoma

(apoptosis)

Decrease

Decrease

2001

Greenberg et al. Cell 1999

Qi et al Can Res 2003T cell lymphoma (ATM-/-)

Hepatocellular carcinoma (Alb-uPA-/-)

Decrease (apoptosis)

Decrease

Qi et al. Can. Res. 2003 Wong et al. Nature 2003

Farazi et al. Can. Res. 2003(Alb uPA / )

Soft tissue sarcoma (p53-/-)

Increase (loss of apoptosis)

Artandi et al. Nature 2000

David FeldserDavid Feldser

Eµ-Myc induced B-cell lymphoma growthEµ Myc induced B cell lymphoma growth can be blocked by short telomeres

Myc+ mTR+ / - mTR+ / -

+ +Myc+ mTR- / - G1 Myc+ G1 G1

Myc+ G2 G2

Myc+ G3 G3

Myc+ G4 G4

+Myc+ G5 G5

Myc+ G6 G6

Feldser & Greider Cancer Cell 2007

y

Short telomeres protect againstShort telomeres protect against B-cell lymphoma

Eμ-mycmTR-/- G6

ent

aliv

e

Eμ-myc - mTR-/-G1

Eμ-mycmTR+/+ mice

Per

cen

G1

DFeldser & Greider Cancer Cell

2007

Days

Both apoptosis and senescence block tumor growth

Short telomere

p53

Bcl‐2Bcl‐2

ApoptosisSenescenceFeldser & Greider Cancer Cell

2007

Nature 2001

Clinical features of dyskeratosis congentiaClinical features of dyskeratosis congentia

• Dermatologic criteriaSkin hyperpigmentation– Skin hyperpigmentation

– Oral leukoplakiaNail dystrophy– Nail dystrophy

• Mortality––Aplastic anemiaAplastic anemia–Pulmonary fibrosis–Cancer

Julian ChenJulian Chen

Telomerase RNA secondary structureTelomerase RNA secondary structureCR4/CR5

Human TelomeraseRNA

TERT

RNA

SnoRNAPseudoknot/Template

Dyskerin

Nop10Nhp2

TERT

Mitchell et al. MCB 1999

Chen et al. Cell 2000

TERT mutations cause autosomalTERT mutations cause autosomal dominant inheritance of dyskeratosis congentiacongentia

Genetic anticipation

Hopkins Family 1I 1 2

Genetic anticipation.

Affected people are1 2 3 4 5 6 7II

Affected people are heterozygous for hTR or hTERT mutations. 1 2 3 4III

?

Dominant negative or h l i ffi i ?haploinsufficiency?

Armanios et al. PNAS (2005)

Cast/Ei mice have short telomeresCast/Ei mice have short telomeres similar to humans

Bred mTR null Bred mTR null allele onto Cast/Ei background

Hemann & Greider NAR 2000

Ling Yang HaoLing-Yang Hao

Progressive telomere shortening duringProgressive telomere shortening during mTR+/- breeding

mTR+ / - mTR+ / -

HG1 HG1

HG2 HG2

HG3 HG3

HG4 HG4

HG5

HG4 HG4

HG5

Short telomeres in CAST/EiJ mTR+/-Short telomeres in CAST/EiJ mTR /mice

Hao et al. Cell 2005

Inheritance of short telomeres decreasesInheritance of short telomeres decreases survival

mTR+ / - mTR+ / -

HG1 HG1

KO(G3)

KO(G2)HG2 HG2

HG3 HG3

KO(G4)

KO(G3)HG3 HG3

HG4 HG4

HG5 KO(G5)

MargaretMargaret Strong

Decreased survival of mTR-/- from laterDecreased survival of mTR / from later generation heterozygous crosses

75

100KO from HG3 parentsKO from HG6 parents

W T

KO from HG8 parentsval

50

75 KO from HG8 parents

ent s

urvi

v

0

25Perc

e

0 100 200 300 4000

Days

Inheritance of short telomeres decreases survival.Resembles genetic anticipation in dyskeratosis congenita. g p y g

Telomere shortening during mTR+/-Telomere shortening during mTR /breeding affects wildtype offspring

mTR+ / - mTR+ / -

HG1 HG1

HG2 HG2

HG3 HG3

HG4 HG4

HG5 KO(G5)WT5*

HG4 HG4

HG5 KO(G5)WT5

Short telomeres in Wt5* miceShort telomeres in Wt5 mice

HG4 HG4

KO HG5 WT5*

Hao et al. Cell 2005

Short telomeres cause phenotypes inShort telomeres cause phenotypes in Wt* mice

Testis Apoptosis

Genetic disease in the absence of telomerase mutation (Occult genetic disease)( g )

Mary ArmaniosMary Armanios

Telomere shortening leads to geneticTelomere shortening leads to genetic anticipation

Hopkins Family 1

I 1 2

Genetic anticipation f lof pulmonary

fibrosis.1 2 3 4 5 6 7II

?

1 2 3 4III

Armanios et al. PNAS (2005)

NEJM 357 p1317 (2007)

Table 2 S ectru of bone arro Table 2 Spectrum of bone marrow, lung, and liver disease seen in individuals with syndromes of ytelomere shortening

Annu. Rev. Genomics Hum. Genet. 2009. 10:45-61

Many phenotypes from telomereMany phenotypes from telomere shortening syndromes share features of age related diseaseage related disease

Bone marrow failure Liver disease Immune senescence Chemotherapy

Increased cancer incidence

Chemotherapy intolerancePulmonary fibrosis Pulmonary fibrosis

•Implies short telomeres may play a role in disease without mutation.

Wildtype mice with short telomeres show a ypdecreased number of B and T lymphocytes

T CellsB Cells T Cells

Armanios et al. AJHG 2009

Decreased CD4/CD8 ratio in mice withDecreased CD4/CD8 ratio in mice with short telomeres is a hallmark of immune senescencesenescence

* **2 0

CD4/CD8RATIO

1.5

2.0

RATIO

1.0

0.5

wt wt9*1 HG90.0

Armanios et al. AJHG 2009

Extensive telomere length heterogeneityExtensive telomere length heterogeneity in the human population

Valdes et al, 2005 Lancet (2005)(2005)

•Combined effect of initial telomere length and environmental history may contribute to age related disease.

Summary and implicationsy p

Telomerase is essential for telomere maintenance.Telomerase is essential for telomere maintenance.

Telomere shortening leads to cell death or senescence after many cell divisions.

Short telomeres inhibit tumor growth through apoptosis or senescence. Telomerase inhibitors may be effective in cancer

therapy.

Haploinsufficiency for telomerase causes telomere Haploinsufficiency for telomerase causes telomere shortening.

Short telomeres limit cell growth even in the gpresence of telomerase. Short telomeres may limit long term stem cell division.

Summary and implicationsy p

I h it f h t t l h t Inheritance of short telomeres causes phenotypes even in wildtype animals (Wt* or occult genetic disease). Short telomeres may cause loss of tissue renewal in

normal aging population.

T l l th di t t f t i Telomere length may predict onset of certain age related diseases.

Telomere shortening during mTR+/-Telomere shortening during mTR /breeding affects wildtype offspring

mTR+ / - mTR+ / -

HG1 HG1

HG2 HG2

HG3 HG3

HG4 HG4

HG5 KO(G5)WT5*

HG4 HG4

HG5 KO(G5)WT5

Decreased survival of mTR+/- from laterDecreased survival of mTR / from later generation heterozygous crosses

Inheritance of short telomeres decreases survival.Resembles genetic anticipation in dyskeratosis congenita. g p y g

Armanios et al. AJHG 2009

Telomere length equlibrium isTelomere length equlibrium is reestablished after five generations

Armanios et al. AJHG (2009)

Wt* mice reestablish telomereWt mice reestablish telomere equilibrium after several generations

mTR+ / - HG4 mTR+ / - HG4

mTR+ / + WT5*1 mTR+ / + WT5*1

mTR+ / + WT5*2 mTR+ / + WT5*2

TR / WT5*3 TR / WT5*3mTR+ / + WT5*3 mTR+ / + WT5*3

mTR+ / + WT5*4 mTR+ / + WT5*4

Armanios et al. AJHG 2009